WO2016107109A1 - 一种胶原蛋白-羟基磷灰石神经支架及其制备方法 - Google Patents

一种胶原蛋白-羟基磷灰石神经支架及其制备方法 Download PDF

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WO2016107109A1
WO2016107109A1 PCT/CN2015/082174 CN2015082174W WO2016107109A1 WO 2016107109 A1 WO2016107109 A1 WO 2016107109A1 CN 2015082174 W CN2015082174 W CN 2015082174W WO 2016107109 A1 WO2016107109 A1 WO 2016107109A1
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collagen
hydroxyapatite
ngf
nerve
stent
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PCT/CN2015/082174
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French (fr)
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李扬德
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东莞颠覆产品设计有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen

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  • the invention relates to the technical field of nerve stents, in particular to a collagen-hydroxyapatite nerve stent and a preparation method thereof.
  • Peripheral nerve damage or breakage due to various trauma causes a decrease or loss of sensory and motor function. Severe peripheral nerve injury often leads to paralysis or permanent loss of labor. Peripheral nerve injury has a high incidence in China. According to statistics, in trauma patients, nerve injury of the extremities accounts for about 10% of the total number of trauma, and about 60% of the fractures of firearm injuries have neurological injuries. To this end, we need to find the ideal treatment for peripheral nerve injury. For peripheral nerve fractures, if the fracture gap is large, the fractured peripheral nerve must be regenerated and repaired by bridging the graft.
  • Stone hydroxyapatite is the main component of vertebrate bones and teeth.
  • the content of hydroxyapatite in human enamel is above 96%. Hydroxyapatite has excellent biocompatibility.
  • the present invention provides a collagen-hydroxyapatite nerve scaffold and a preparation method thereof.
  • the neural scaffold prepared by the method has good mechanical properties, is biodegradable, and has a three-dimensional structure required for nerve regeneration.
  • NGF has far-reaching significance for nerve repair.
  • the technical scheme of the present invention is as follows: a collagen-hydroxyapatite nerve scaffold comprising a scaffold film and a degradable metal wire wrapped by a scaffold film, the main component of which is collagen, nerve growth factor ( NGF) and hydroxyapatite.
  • the NGF and gelatin are made into NGF gelatin microspheres distributed in the scaffold film, and the mass content of the NGF gelatin microspheres is 20-40% of the total mass content of the PLGA powder and the collagen powder.
  • the stent is in the form of a sheet, a strip or a cylinder.
  • the degradable wires are distributed along the longitudinal and transverse directions of the stent.
  • the main component of the degradable metal wire is magnesium, zinc, calcium, iron or an alloy thereof.
  • the preparation method of the collagen-hydroxyapatite nerve scaffold comprises the following steps:
  • step 2) Preparation of composite material: Weigh the hydroxyapatite powder and the collagen powder obtained in step 1) according to the mass ratio of 8-6:2-4, add it to distilled water, adjust the pH value of the solution to 8-9, and use a magnetic stirrer. Stirring was heated to 36-39 ° C, stirring was continued for 8-10 h, and dried under vacuum to obtain a composite powder;
  • NGF gelatin microspheres were prepared by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro. Ball
  • preparing a stent film material completely dissolving the composite powder in dichloromethane, adding the NGF gelatin microspheres obtained in the step 3), and uniformly dispersing ultrasonically to obtain a stent film material;
  • the scaffold film obtained in the step 4) into the mold cavity of the film forming mold, and placing the straightened degradable metal wire in the transverse direction and the longitudinal direction of the stent film in the molding direction of the stent film, so that the degradable metal wire is immersed in the stent film
  • the solvent is naturally volatilized and dried, and vacuum is applied for 48 hours. At this time, the dried stent film is wrapped around the degradable metal wire to obtain the nerve stent.
  • the particle size of the NGF gelatin microspheres was 1-20 ⁇ m, and the sealing ratio of the microspheres to NGF was 81.5%.
  • the NGF gelatin microspheres are used in an amount of 20-40% of the total amount of the collagen-hydroxyapatite composite.
  • the main component is collagen, which also contains fibronectin, laminin, aminodextran, proteoglycan, etc., which are beneficial to nerve conduction. And the growth of axons, with good biocompatibility.
  • the use of ECM alone for nerve injury transplantation will make it difficult to provide morphological support for nerve growth because ECM is too soft, and at the same time, these ECMs will be calcified in a short time. Therefore, the combination of collagen and stone hydroxyapatite makes the prepared scaffold have certain mechanical properties, can provide morphological support for nerve growth, and also prevent collagen calcification.
  • scanning electron fiber mirrors show that the internal structure of the obtained artificial nerve scaffold is honeycomb, which provides space for the nerve cells to crawl and grow.
  • Degradable metals and their alloys can guide the growth of nerve cells, which is beneficial to the repair of nerve cells.
  • NGF is a biologically active substance. During the process of peripheral nerve repair, nerve tissue secretes NGF. Adding exogenous NGF is beneficial to nerve repair.
  • NGF gelatin microspheres are prepared by using gelatin. Since gelatin is a water-soluble substance, it is not dissolved in an organic solvent. In the preparation of the material, gelatin can isolate NGF from the organic solvent, thereby facilitating the maintenance of NGF activity. Gelatin also contributes to the slow release of the NGF.
  • Stone hydroxyapatite is the main component of vertebrate bones and teeth.
  • the content of hydroxyapatite in human enamel is above 96%. Hydroxyapatite has excellent biocompatibility.
  • the addition of stone hydroxyapatite to the nerve scaffold can impart good mechanical properties to the stent.
  • the collagen-hydroxyapatite nerve scaffold of the invention has good mechanical properties, biodegradability, and has the three-dimensional structure and NGF required for nerve regeneration, and has profound significance for nerve repair.
  • FIG. 1 is a schematic view showing the structure of a collagen-hydroxyapatite nerve scaffold according to the present invention.
  • Fig. 2 is a three-dimensional structure of a porous structure formed by decellularization of pig skin in the present invention.
  • Fig. 3 is a view showing the morphology of the NGF gelatin microspheres obtained in the present invention under a microscope.
  • a collagen-hydroxyapatite nerve stent comprises a stent film 1 and a degradable metal wire 2 wrapped by a stent film 1.
  • the degradable metal wire 2 is distributed along the lateral and longitudinal directions of the nerve stent.
  • the stent can be prepared into various shapes according to the needs of use, and is generally cylindrical or sheet-shaped.
  • the main material of the stent film 1 is a collagen-hydroxyapatite composite material; the mass content of the NGF gelatin microspheres is 20-40% of the total mass content of the collagen-hydroxyapatite composite material.
  • the main component of the degradable metal wire 2 is magnesium, zinc, calcium, iron or an alloy thereof.
  • Example 1 The preparation method of the collagen-hydroxyapatite nerve scaffold comprises the following steps:
  • the resulting collagen powder was observed by H.E staining and scanning electron microscopy, as shown in FIG. It can be seen that the pig skin is decellularized to form a loose porous three-dimensional structure.
  • step 2) Preparation of composite material: Weigh the hydroxyapatite powder and the collagen powder obtained in step 1) according to the mass ratio of 8-6:2-4, add it to distilled water, adjust the pH value of the solution to 8-9, and use a magnetic stirrer. Stirring was heated to 36-39 ° C, stirring was continued for 8-10 h, and dried under vacuum to obtain a composite powder;
  • NGF gelatin microspheres were prepared by a 20% aqueous gelatin solution, the concentration of NGF was 0.06-0.10 ⁇ g/ml, and the mixed solution was added to a salad oil of 3.5 times its volume, and preheated to 37 ° C.
  • the oil-water emulsion was emulsified by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro.
  • the shape of the obtained NGF gelatin microspheres was observed by a microscope, as shown in FIG.
  • the NGF gelatin microspheres have a particle diameter of 1-20 ⁇ m.
  • the composite powder is completely dissolved in dichloromethane, and the NGF gelatin microspheres obtained in the step 3) are added, and the ultrasonic dispersion is uniform to obtain a stent film; the amount of the NGF gelatin microspheres is collagen- 20-40% of the total amount of hydroxyapatite composite;
  • the scaffold film obtained in the step 4) into the mold cavity of the film forming mold, and placing the straightened degradable metal wire in the mold cavity along the transverse direction and the longitudinal direction of the stent film forming direction, so that the degradable metal wire is immersed in the bracket In the film, the solvent is naturally volatilized and dried, and vacuum is applied for 48 hours. At this time, the dried stent film is wrapped around the degradable metal wire to obtain the nerve support.
  • the cross section of the obtained nerve scaffold was observed by scanning electron microscopy, and the obtained artificial nerve scaffold showed a honeycomb structure in a cross section.
  • the honeycomb structure provides the required voids for the creep and extension of nerve cells.
  • Example 2 The preparation method of the collagen-hydroxyapatite nerve scaffold comprises the following steps:
  • the obtained collagen powder was observed by H.E staining and scanning electron microscopy. It was observed that the pig skin was decellularized to form a porous three-dimensional structure.
  • step 2) Preparation of composite material: Weigh the hydroxyapatite powder and the collagen powder obtained in step 1) according to the mass ratio of 8-6:2-4, add it to distilled water, adjust the pH value of the solution to 8-9, and use a magnetic stirrer. Stirring was heated to 36-39 ° C, stirring was continued for 8-10 h, and dried under vacuum to obtain a composite powder;
  • NGF gelatin microspheres were prepared by stirring at 10000 rpm for 10 min, frozen to -20 ° C, and the emulsion was taken out and centrifuged at 2000 rpm for 10 min. The precipitate was taken and washed with acetone for 3-5 times. The precipitate after washing was NGF gelatin micro.
  • the ball; the NGF gelatin microspheres have a particle diameter of 1-20 ⁇ m.
  • the composite powder is completely dissolved in dichloromethane, and the NGF gelatin microspheres obtained in the step 3) are added, and the ultrasonic dispersion is uniform to obtain a stent film; the amount of the NGF gelatin microspheres is collagen- 20-40% of the total amount of hydroxyapatite composite;
  • the scaffold film obtained in the step 4) into the mold cavity of the film forming mold, and placing the straightened degradable metal wire in the transverse direction and the longitudinal direction of the stent film in the molding direction of the stent film, so that the degradable metal wire is immersed in the stent film
  • the solvent is naturally volatilized and dried, and vacuum is applied for 48 hours. At this time, the dried stent film is wrapped around the degradable metal wire to obtain the nerve stent.
  • the cross section of the artificial nerve stent obtained by scanning electron microscopy showed that the artificial nerve stent obtained had a honeycomb structure in cross section.
  • the honeycomb structure provides the required voids for the creep and extension of nerve cells.

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Abstract

一种胶原蛋白-羟基磷灰石神经支架，包括支架膜料（1）以及支架膜料（1）包裹的可降解金属丝（2），所述支架膜料（1）的主要成分为胶原蛋白，神经生长因子(NGF)和羟基磷灰石。该方法制备的人工神经支架力学性能良好，生物可降解，具有神经再生所需要的三维结构，还可以对NGF缓慢释放，对神经修复具有深远的意义。

Description

一种胶原蛋白-羟基磷灰石神经支架及其制备方法 技术领域
本发明涉及神经支架技术领域，具体地，涉及一种胶原蛋白-羟基磷灰石神经支架及其制备方法。
背景技术
由于各种外伤原因导致的周围神经损伤或断裂造成伤者感觉和运动功能的下降或丧失。严重的外周神经损伤往往导致患者瘫痪或永久丧失劳动力。外周神经损伤在我国发病率较高，据统计，在外伤患者中，四肢神经伤约占外伤总数的10％，火器伤骨折中约有60％的合并神经伤。为此，需寻找理想的外周神经伤治疗手段。对于外周神经断裂类损伤，若断裂缺口较大，就须借助对缺口进行桥接移植手术才能使断裂的周围神经得到再生修复。
目前，常用的移植体有自体移植体和人工神经支架。自体移植体来源有限且会带来供体后遗症。人工神经支架方面，由于非降解导管材料可能导致神经压迫和二次手术的风险，近年来研究的主要趋向于生物可降解支架材料的开发和研究。当前，美国已经批准多种品牌，如，NeuraGen、SaluBridge，Neurolac等的神经导管用于临床。然而，我国在神经支架方面的研究较为落后，并没有开发出具有自主知识产权的神经导管。
石羟基磷灰石(HAP)是脊椎动物骨骼和牙齿的主要组成，人的牙釉质中羟基磷灰石的含量在96％以上。羟基磷灰石具有优良的生物相容性。
发明内容
为了克服现有技术的不足，本发明提供了一种胶原蛋白-羟基磷灰石神经支架及其制备方法，该方法制备的神经支架力学性能良好，生物可降解，具有神经再生所需要的三维结构和NGF，对神经修复具有深远的意义。
本发明的技术方案如下：一种胶原蛋白-羟基磷灰石神经支架，包括支架膜料以及支架膜料包裹的可降解金属丝，所述支架膜料的主要成分为胶原蛋白，神经生长因子(NGF)和羟基磷灰石。
所述NGF与明胶制成NGF明胶微球分布与所述支架膜料中，所述NGF明胶微球的质量含量为PLGA粉末和胶原蛋白粉末总质量含量的20-40％。
所述支架为片状，长条状或圆柱状。
所述可降解金属丝沿所述支架的纵向和横向分布。
所述可降解金属丝的主要成分为镁，锌，钙，铁或其合金。
所述胶原蛋白-羟基磷灰石神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂18-36h；用清水漂洗三次，每次18-24h；于0.5％的胰蛋白酶中在37℃振荡浸泡18-30h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48-72h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白；
2)复合材料的制备：按质量比例8-6∶2-4称量羟基磷灰石粉末和步骤1)所得胶原蛋白粉末，加入蒸馏水中，调节溶液pH值为8-9，用磁力搅拌器搅拌经加热至36-39℃，继续搅拌8-10h，真空干燥，得到复合物粉末；
3)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；
4)制备支架膜料：将复合物粉末完全溶解在二氯甲烷中，加入步骤3)所得NGF明胶微球，超声分散均匀，得到支架膜料；
5)将步骤4)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向的横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述神经支架。
所述NGF明胶微球的粒经为1-20μm，微球对NGF的封载率为81.5％。
所述NGF明胶微球的用量为胶原蛋白-羟基磷灰石复合材料总量的20-40％。
动物皮被处理后，脱出了细胞，形成了结构松散的ECM，其主要成分是胶原蛋白，还含有纤维连接蛋白，层粘连接蛋白，氨基葡聚糖，蛋白聚糖等，这些有利于神经传导以及轴突的生长，具有良好的生物相容性。将ECM单独用于神经损伤移植，会由于ECM太软，难以为神经生长提供形态学上的支撑，同时，短时间内，这些ECM就会被钙化。因此，将胶原蛋白与石羟基磷灰石结合使用使得制成的支架具有一定的力学性能，能够为神经生长提供形态学上的支撑，也防止胶原蛋白钙化。另外，扫描电子纤维镜表明，所得人工神经支架的内部结构为蜂窝状，为神经细胞的爬行和生长提供了空间。
可降解金属及其合金可以引导神经细胞生长，从而有利于神经细胞的修复。
NGF是生物活性物质，在周围神经修复的过程中，神经组织会分泌NGF，添加外源的NGF有利于神经的修复。本发明中，使用明胶制备NGF明胶微球，由于明胶是水溶性物质，在有机溶剂中不溶解，在材料的制备中明胶可将NGF与有机溶剂隔离，从而有利于保持NGF活性。明胶还有助于所述NGF的缓慢释放。
石羟基磷灰石(HAP)是脊椎动物骨骼和牙齿的主要组成，人的牙釉质中羟基磷灰石的含量在96％以上。羟基磷灰石具有优良的生物相容性。神经支架中加入石羟基磷灰石可以赋予支架良好的力学性能。
本发明的有益效果为：本发明所述胶原蛋白-羟基磷灰石神经支架力学性能良好，生物可降解，具有神经再生所需要的三维结构和NGF，对神经修复具有深远的意义。
附图说明：
图1为本发明所述胶原蛋白-羟基磷灰石神经支架的结构示意图。
图2为本发明中猪皮经脱细胞处理后形成的疏松多孔的三维结构。
图3为显微镜下本发明中所得NGF明胶微球的形态。
具体实施方式
下面结合附图和优选实施例对本发明作进一步的描述，但本发明的实施方式不限于此。
参照图1，一种胶原蛋白-羟基磷灰石神经支架，包括支架膜料1以及支架膜料1包裹的可降解金属丝2。所述可降解金属丝2沿神经支架的横向和纵向分布。所述支架可以根据使用需要制备成各种形状，一般为圆筒形，也可以是片状。
所述支架膜料1主料为胶原蛋白-羟基磷灰石复合材料；所述NGF明胶微球的质量含量为胶原蛋白-羟基磷灰石复合材料总质量含量的20-40％。
所述可降解金属丝2的主要成分为镁，锌，钙，铁或其合金。
实施例1：所述胶原蛋白-羟基磷灰石神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂24h；用清水漂洗三次，每次20h；于0.5％的胰蛋白酶中在37℃振荡浸泡124h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡56h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白；
用H.E染色及扫描电镜观测所得胶原蛋白粉末，如图2所示。经观察可知，猪皮经脱细胞处理后形成疏松多孔的三维结构。
2)复合材料的制备：按质量比例8-6∶2-4称量羟基磷灰石粉末和步骤1)所得胶原蛋白粉末，加入蒸馏水中，调节溶液pH值为8-9，用磁力搅拌器搅拌经加热至36-39℃，继续搅拌8-10h，真空干燥，得到复合物粉末；
3)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；用显微镜观察所得NGF明胶微球的形态，如图3所示。所述NGF明胶微球的粒经为1-20μm。
4)制备支架膜料：将复合物粉末完全溶解在二氯甲烷中，加入步骤3)所得NGF明胶微球，超声分散均匀，得到支架膜料；所述NGF明胶微球的用量为胶原蛋白-羟基磷灰石复合材料总量的20-40％；
5)将步骤4)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向的横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架 膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述神经支架。
用扫描电镜观测所得神经支架的横断面，显示所得人工神经支架的横断面呈蜂窝状结构。蜂窝状结构为神经细胞其中的爬行及延伸提供了需要的空隙。
实施例2：所述胶原蛋白-羟基磷灰石神经支架的制备方法，包括如下步骤：
1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂36h；用清水漂洗三次，每次18h；于0.5％的胰蛋白酶中在37℃振荡浸泡18h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48-72h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白；
用H.E染色及扫描电镜观测所得胶原蛋白粉末，经观察可知，猪皮经脱细胞处理后形成疏松多孔的三维结构。
2)复合材料的制备：按质量比例8-6∶2-4称量羟基磷灰石粉末和步骤1)所得胶原蛋白粉末，加入蒸馏水中，调节溶液pH值为8-9，用磁力搅拌器搅拌经加热至36-39℃，继续搅拌8-10h，真空干燥，得到复合物粉末；
3)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；所述NGF明胶微球的粒经为1-20μm。
4)制备支架膜料：将复合物粉末完全溶解在二氯甲烷中，加入步骤3)所得NGF明胶微球，超声分散均匀，得到支架膜料；所述NGF明胶微球的用量为胶原蛋白-羟基磷灰石复合材料总量的20-40％；
5)将步骤4)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向的横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述神经支架。
用扫描电镜观测所得人工神经支架的横断面显示所得人工神经支架的横断面呈蜂窝状结构。蜂窝状结构为神经细胞其中的爬行及延伸提供了需要的空隙。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其架构形式能够灵活多变，可以派生系列产品。只是做出若干简单推演或替换，都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。

Claims (9)

  1. 一种胶原蛋白-羟基磷灰石神经支架，其特征在于，包括支架膜料以及支架膜料包裹的可降解金属丝，所述支架膜料的主要成分为胶原蛋白，神经生长因子(NGF)和羟基磷灰石。
  2. 如权利要求1所述的胶原蛋白-羟基磷灰石神经支架，其特征在于，所述羟基磷灰石和所述胶原蛋白的质量比为8-6∶2-4。
  3. 如权利要求1所述的胶原蛋白-羟基磷灰石神经支架，其特征在于，所述NGF与明胶制成NGF明胶微球分布与所述支架膜料中，所述NGF明胶微球的质量含量为所述羟基磷灰石和所述胶原蛋白总质量含量的20-40％。
  4. 如权利要求1所述的胶原蛋白-羟基磷灰石神经支架，其特征在于，所述支架为片状，长条状或圆柱状。
  5. 如权利要求1所述的胶原蛋白-羟基磷灰石神经支架，其特征在于，所述可降解金属丝沿所述支架的纵向和横向分布。
  6. 如权利要求1所述的胶原蛋白-羟基磷灰石神经支架，其特征在于，所述可降解金属丝的主要成分为镁，锌，钙，铁或其合金。
  7. 如权利要求1-6任一项所述的胶原蛋白-羟基磷灰石神经支架的制备方法，其特征在于，包括如下步骤：
    1)胶原蛋白粉末的制备：动物皮去除皮下脂肪组织后，洗净，切碎，在碱性溶液中脱脂18-36h；用清水漂洗三次，每次18-24h；于0.5％的胰蛋白酶中在37℃振荡浸泡18-30h；在10mMTrisHCl(pH8.0)+2％TritonX-100中在37℃振荡浸泡48-72h；室温下用清水清洗2d；取出用灭菌的PBS浸泡，冷冻干燥后经机械粉碎，过400目分子筛，得到胶原蛋白粉末；所述胶原蛋白粉末的主要成分为胶原蛋白，还含有纤维连接蛋白和层粘连蛋白；
    2)复合材料的制备：按质量比例8-6∶2-4称量羟基磷灰石粉末和步骤1)所得胶原蛋白粉末，加入蒸馏水中，调节溶液pH值为8-9，用磁力搅拌器搅拌经加热至36-39℃，继续搅拌8-10h，真空干燥，得到复合物粉末；
    3)NGF明胶微球的制备：在20％的明胶水溶液中加入NGF，所述NGF浓度为0.06-0.10μg/ml，将混合溶液加入要其体积3.5倍的色拉油中，预热到37℃以10000rpm搅拌10min乳化形成油水乳液，冷冻至-20℃，取出乳液放入冷冻离心机中以2000rpm离心10min，取沉淀物，用丙酮洗涤3-5次，洗涤后的沉淀物即为NGF明胶微球；
    4)制备支架膜料：将复合物粉末完全溶解在二氯甲烷中，加入步骤3)所得NGF明胶微球，超声分散均匀，得到支架膜料；
    5)将步骤4)所得支架膜料转入制膜模具的模具腔中，模具腔中沿支架膜料成型方向的横向和纵向放置拉直的可降解金属丝，使可降解金属丝浸入支架膜料中，溶剂自然挥发干燥，抽真空48h，此时，干燥的支架膜料包裹所述可降解金属丝，得到所述神经支架。
  8. 如权利要求7所述的胶原蛋白-羟基磷灰石神经支架的制备方法，其特征在于，所述NGF明胶微球的粒经为1-20μm，微球对NGF的封载率为81.5％。
  9. 如权利要求7所述的胶原蛋白-羟基磷灰石神经支架的制备方法，其特征在于，所述NGF明胶微球的用量为羟基磷灰石和胶原蛋白总量的20-40％。
PCT/CN2015/082174 2014-12-29 2015-06-24 一种胶原蛋白-羟基磷灰石神经支架及其制备方法 WO2016107109A1 (zh)

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